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doi: 10.1242/10.1242/dev.00152

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Cellularisation in the endosperm of Arabidopsis thaliana is coupled to mitosis and shares multiple components with cytokinesis

¹ Laboratoire de Reproduction et Développement des Plantes, UMR 5667, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, F-69364 Lyon, Cedex 07, France
² Plant Research Department, PRD-301, Risø National Laboratory, PO Box 49, DK-4000 Roskilde, Denmark
³ ZMBP — Center of Plant Molecular Biology, Developmental Genetics, University of Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen, Germany
⁴ Carnegie Institution of Washington, Department of Plant Biology, 260 Panama Street, Stanford, California 94305, USA
⁵ Laboratoire de Biologie des Semences, UMR INRA/INA-PG, Route de Saint-Cyr, 78026 Versailles Cedex, France

View larger version (124K): [in a new window]   Fig. 4. FASS/TON2 is required for correct periclinal divisions in the peripheral endosperm. Confocal sections of seeds from heterozygous fass/ton2 parent plants. (A) Wild-type reference seed with torpedo stage embryo and cellularised endosperm. (B) fass/ton2 mutant seed from same silique as A. (C,D) Details of first periclinal division in the PEN of wild type (C) and fass/ton2 (D). (E) Absence of thin outer cell layer in cellularised PEN in fass/ton2. Scale bars: 100 µm (A,B) and 25 µm (C,D,E).

View larger version (160K): [in a new window]   Fig. 1. Initiation of cellularisation is coupled to mitosis. (A-F) Time series visualising nuclear division and cellularisation in the PEN (see movie: http://dev.biologists.org/supplemental/). The in vivo progression of cellularisation was monitored by time-lapse confocal microscopy of living seeds from the GFP-expressing line KS22. GFP accumulates at sites of forming cell walls between sister nuclei (arrowheads). This series is part of an 8 hour recording of endosperm development. Frames were acquired every 10 minutes. Images in A and F mark the beginning and end of the series, respectively, and were recorded with a decreased acquisition rate to give better resolution. 200 minutes after the beginning of recording, a mitotic wave was initiated at the anterior pole (upper side of the section) and crossed the PEN (B,C). Immediately after mitosis cell plates are visible between sister nuclei (D, arrowhead) and slightly later between non-sister nuclei (E, arrowhead). Cellularisation appears to be complete 6 hours after the mitotic wave (F). (G-J) Detail of the formation of a cell plate between sister nuclei following the 8th mitotic cycle in the PEN observed in fixed material. (G) Metaphase, (H) anaphase, (I) telophase, and (J) the clear formation of a cell plate. Scale bar: 20 µm (A-F); 5 µm (G-J).

View larger version (82K): [in a new window]   Fig. 2. Comparison of the effect of knolle and keule on cytokinesis and on the cellularisation in the endosperm. Seeds with embryos at late heart stage originate from heterozygous mutant plants. Homozygous mutant seeds are identified by their embryo phenotype and seeds with a wild-type phenotype from the same silique are used as controls. (A,B) Wild-type reference seed with late heart stage embryo and fully cellularised endosperm around the embryo (A) and in the PEN (B). (C,D) keule mutant seeds contain an embryo with multiple defect in cytokinesis, and multinucleate enlarged cells (C) whereas the endosperm cellularisation is not affected (D). (E,F) knolle produces seeds with embryos defective in cytokinesis (E) and non-cellularised endosperm (F). Embryos of the double mutant knolle/keule are characterised by a complete absence of cytokinesis and are reduced to multinucleated tubes (G). However the defect of cytokinesis in the endosperm is no more pronounced than in knolle (G,H). Scale bars represent 20 µm.

View larger version (83K): [in a new window]   Fig. 3. Cytokinesis-related genes are required for endosperm cellularisation. (A,C,E,G) Both the embryo and the MCE, have defective cytokinesis in the mutants hinkel (A) open house (C) runkel (E) and pleiade (G). (B,D,F,H) Corresponding confocal sections of the PEN with obvious defects in cellularisation. Scale bars: 20 µm.

View larger version (138K): [in a new window]   Fig. 5. The SPÄTZLE gene is required for endosperm cellularisation, but not for cytokinesis in the embryo. (A,B) The spätzle embryo does not show any morphogenetic defect (A, heart stage, B, torpedo stage) although it is surrounded by non cellularised MCE. (C) The PEN does not undergo cellularisation during the heart stage. However division of nuclei is maintained but is not always followed by proper separation of NCDs (D,E) and in some cases nuclei remain attached by incompletely separated nuclear envelopes (E, arrowhead). Hence multinucleated NCDs form and nuclei fuse (F) leading to large nuclei that display multiple nucleoli (G, arrowheads). Scale bars represent 20 µm.

View larger version (134K): [in a new window]   Fig. 6. Anteroposterior polarity in cellularisation-defective mutants. (A,C,E) Cross sections of the posterior most part of the endosperm. In the wild-type the posterior-most part of the endosperm does not cellularise. (A) This part remains syncytial and contains two types of multinucleated large NCDs, the nodules (n) and the cyst (c). (C,E) In both knolle and spätzle backgrounds nodules and cysts are observed at the posterior pole similar to that in the wild type (early torpedo stage). In spätzle endosperm (E), the coalescence of NCDs is observed in the non-cellularised PEN and its distinction from the CZE is less clear than in the wild type or in knolle. (B,D,F) GFP expression in the enhancer trap line KS117. (B) After the globular stage, the posterior pole remains the only site where the GFP marker is expressed (Sørensen et al., 2001). (D,F) However, the expression of the marker KS117 is confined to the posterior pole as in the wild type in both knolle/+ (D) and spätzle/spätzle (F) backgrounds. Scale bar: 20 µm.